Polymerized hemoglobin solutions having reduced amounts of tetramer and method for preparing

ABSTRACT

A substantially tetramer free hemoglobin solution and a method for producing a substantially tetramer free hemoglobin solution. The method includes polymerizing a solution of hemoglobin, treating the polymerized hemoglobin solution to partially degrade the polymer to tetramer and removing tetramer from the hemoglobin solution.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial. No. 60/443,436 filed Jan. 29, 2003.

FIELD OF THE INVENTION

[0002] The invention relates to stabilized oxygen carrying solutions.More specifically, it relates to hemoglobin solutions that have beentreated to enhance polymer bond stability and to remove tetramer thathas been elaborated.

BACKGROUND OF THE INVENTION

[0003] There is a consistent need for ready blood products for anever-increasing surgical and trauma load, and to supplement blood bankshortages. Oxygen carrying solutions, such as hemoglobin-derivedsolutions can be used in place of whole blood or red blood cells forpatients having a need for augmented oxygen carrying capacity. Becausethey are not dependent upon donor availability, such solutions can bemade readily available in an emergency situation or during a blood bankshortage. Also, due to risk of infection of blood borne pathogens as aresult of a blood transfusion, a patient may prefer a hemoglobin-derivedsolution for transfusion in place of whole blood or red blood cells. Inparticular, such solutions may include, but are not limited to, oxygencarriers, blood substitutes, and hemoglobin-derived compositions such asthose disclosed in U.S. Pat. Nos. 6,498,141, 6,133,425, 5,464,814,5,438,041, 5,217,648, 5,194,590, 5,061,688, and 4,826,811, the teachingsof which are incorporated herein by reference in their entirety.

[0004] Stroma-free hemoglobin is known in the art to have oxygentransport and reversible oxygen (or ligand) binding capacities. However,hemoglobin solutions, while capable of carrying sufficient quantities ofoxygen to support life, have presented challenges because of severalundesirable side effects such as a decrease in kidney performance. Theseeffects were thought to be due to the presence of unwanted contaminantssuch as bacterial endotoxin or fragments of red cell membranes (stroma)that is not removed from solution. While contaminants such as these canindeed produce renal alterations, hemoglobin solutions essentially freeof such contaminants still produce substantial renal dysfunction. Thecause for the renal dysfunction can be ascribed to, among other things,physiologically unacceptable amounts of unpolymerized hemoglobintetramer.

[0005] Essentially tetramer free hemoglobin solutions can be used toreplenish essentially all of a human patient's blood volume withoutcausing vasoconstriction, renal toxicity, hemoglobinuria or otherproblems associated with intravenous administration of synthetic orsemisynthetic oxygen carriers and blood substitutes. While thesesolutions provide superior efficacy, the shelf life of the product islimited since the hemoglobin polymer is known to slowly degrade totetrameric units over time. Accordingly, what is needed is a method formaintaining the solution as a substantially tetramer free solution foran extended period to increase the shelf life of the solution.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides a method for producing asubstantially tetramer free hemoglobin solution comprising polymerizinghemoglobin in solution, treating the polymerized hemoglobin to elaboratetetramer and removing tetramer from the polymerized hemoglobin solution.

[0007] In another aspect, the invention relates to a substantiallytetramer free hemoglobin solution produced by polymerizing a solution ofhemoglobin, heat treating the polymerized hemoglobin solution toelaborate tetramer, and removing tetramer from the polymerizedhemoglobin solution.

[0008] In a further aspect, the invention relates to a method forstabilizing a polymerized hemoglobin solution comprising treating thepolymerized hemoglobin solution to partially degrade the polymerizedhemoglobin to tetramer and removing tetramer from the solution.

[0009] In yet another aspect, the invention provides a method forproducing a stabilized, polymerized hemoglobin solution. The methodincludes producing a polymerized hemoglobin solution, removing tetramerfrom the polymerized hemoglobin solution to produce a substantiallytetramer free polymerized hemoglobin solution, aging the polymerizedhemoglobin solution to allow tetramer to elaborate, and removing theelaborated tetramer. The aging may include storing the hemoglobinsolution until the tetramer concentration is greater than about 1.0% oftotal hemoglobin, such as longer than one year.

[0010] The hemoglobin may be derived from mammalian blood, such as humanor bovine blood. The hemoglobin may be polymerized with glutaraldehyde.The tetramer may be removed by filtration. The treatment of thepolymerized solution to elaborate tetramer may be accomplished byheating the solution above about 45° C. for at least about 24 hours. Thetetramer concentration after removing tetramer may be less than about1.0% of total hemoglobin in the solution, or less than about 0.3% oftotal hemoglobin in the solution.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 shows the amount of tetramer elaboration in hemoglobinsolutions stored at 2-8° C.

[0012]FIG. 2 shows the amount of tetramer elaboration in hemoglobinsolutions stored at 23-27° C.

[0013]FIG. 3 shows a comparison of tetramer elaboration between newlyprocessed hemoglobin solutions and reprocessed aged hemoglobin solutionsstored at 2-8° C.

[0014]FIG. 4 shows a comparison of tetramer elaboration between newlyprocessed hemoglobin solutions and reprocessed aged hemoglobin solutionsstored at 23-27° C.

[0015]FIG. 5 shows a comparison of tetramer elaboration between newlyprocessed hemoglobin solutions and hot quenched solutions stored at 2-8°C.

[0016]FIG. 6 shows a comparison of tetramer elaboration between newlyprocessed hemoglobin solutions and hot quenched solutions stored at23-27° C.

[0017]FIG. 7 shows a comparison of tetramer elaboration between newlyprocessed hemoglobin solutions and pre-elaborated solutions stored at2-8° C.

[0018]FIG. 8 shows a comparison of tetramer elaboration between newlyprocessed hemoglobin solutions and pre-elaborated solutions stored at23-27° C.

[0019]FIG. 9 is an HPLC tracing of a purified, polymerized hemoglobinsolution. Polymerized hemoglobin is indicated by the peaks at RT 15.19,16.01, 17.16 and 18.79. Tetramer is indicated by peaks at RT 21.22 and21.83.

[0020]FIG. 10 is an HPLC tracing of a polymerized hemoglobin solutionafter glycine treatment but prior to purification. Polymerizedhemoglobin is indicated by peaks at retention times (RT) 14.62, 15.44,16.60, and 18.24. Tetramer is indicated by the peak at RT 20.71. Polymeris about 75% of this material.

[0021]FIG. 11 is a schematic diagram depicting the portion of theprocess and equipment used to produce a deoxygenated hemoglobin solutionprepared for pyridoxylation and polymerization.

[0022]FIG. 12 is a schematic diagram depicting the portion of theprocess and apparatus to produce, beginning with pyridoxylation andpolymerization, a deoxygenated, purified, pyridoxylated, polymerizedhemoglobin product, and the portion of the process and apparatus forformulating the final hemoglobin product having physiological levels ofelectrolytes.

[0023]FIG. 13 is a schematic diagram depicting a column chromatographypurification process employed in the invention.

[0024]FIG. 14 is a schematic diagram depicting a membrane filtrationpurification process employed in the invention.

DETAILED DESCRIPTION

[0025] The invention provides an oxygen carrying solution comprising anessentially tetramer-free, cross-linked, polymerized, hemoglobin whichis substantially free of stroma, stromal contaminants and othercontaminants.

[0026] Before describing the present invention in detail, a number ofterms will be defined. It is to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting. As used herein, the singular forms “a,”“an,” and “the” include plural referents unless the context clearlydictates otherwise.

[0027] “Hemoglobin” refers to hemoglobin derived from any source.Hemoglobin may be derived from mammals including humans, cattle, pigs,and sheep, or from other sources such as transgenically-producedhemoglobin described in BIO/TECHNOLOGY, 12: 55-59 (1994), andrecombinantly produced hemoglobin, such as the recombinantly producedhemoglobin described in Nature, 356: 258-260 (1992). As used herein, %total hemoglobin (THb) is defined as grams of hemoglobin/100 mL ofsolution.

[0028] A “solution of hemoglobin” refers to a solution of tetrameric orpolymerized hemoglobin molecules where the molecules are not containedwithin a red blood cell. Such a solution need not be free of orsubstantially free of red blood cell stroma or stromal contaminants.However, in one aspect of the invention, solutions of polymerizedhemoglobin are substantially free of red blood cell stroma and stromalcontaminants.

[0029] “Cross-linked” means the chemical placement of molecular“bridges” onto or into a molecule, or between molecules with the purposeof altering the shape, size, function or physical characteristics of themolecule. Cross-linked molecules may be polymerized or non-polymerized,i.e., cross-linked molecules may be tetrameric.

[0030] “Tetramer” or “tetrameric” refers to hemoglobin molecules havinga molecular weight of about 64 Kd; that is, the term refers to bothnative and intramolecularly crosslinked hemoglobin molecules. As usedherein % tetramer refers to the amount of tetramer as a percentage ofthe amount of total hemoglobin (THb) in solution. For example, a 100 mLhemoglobin solution having 10% THb and 1% tetramer has 0.1 g tetramer insolution.

[0031] “Essentially tetramer free” denotes the level of purity withrespect to tetramer contamination at which certain biological responsesto tetramer administered into a mammal are no longer present. A maincriterion is the absence of alterations in renal function whenpharmaceutically effective amounts are infused, that is, at a level ofpurity of about 99% or better (less than about 1% of tetramer ispresent). The preferred product produced by the inventive processcontains no more than about 0.8% tetramer based on the weight of totalhemoglobin (THb). In other words, an essentially tetramer-free productaccording to the invention contains no more then physiologicallyacceptable amounts of unpolymerized hemoglobin tetramer. Particularlypreferred products of the invention contain less than about 0.5%tetramer; the most particularly preferred products of the inventioncontain about 0.3-0.4% tetramer. Such amounts of tetramer have beenfound to be physiologically acceptable.

[0032] “Elaboration” or “tetramer elaboration” refers to an increase ofthe amount of tetramer in solution due to the degradation of polymerizedhemoglobin to tetramer. Degradation of the polymer can be the result oftreatment with chemicals, temperature, time or a combination thereof.Generally, tetramer elaborates upon heating or as the polymerizedsolution ages. Thus, percent tetramer increases during storage of thesolution. Tetramer elaboration can be accelerated by heating thepolymerized hemoglobin solution. Elaboration is said to have occurredafter any increase in the amount of tetramer in solution. As usedherein, the partial degradation of a polymerized hemoglobin refers tosome, but not all, of the polymer in solution being degraded totetramer.

[0033] “Aging” a hemoglobin solution refers to storing the solution forany amount of time at any temperature. Higher temperatures acceleratethe effects of aging on the hemoglobin solution. An “aged” hemoglobinsolution has been stored so that tetramer has elaborated.

[0034] “Pre-elaboration” or “tetramer pre-elaboration” refers to atechnique that utilizes heat treatment to promote tetramer elaboration.

[0035] “Hot quench” refers to a processing technique that involvesheating the solution during the polymerization quench reaction to drivethe reaction to completion.

[0036] “Polymerizing” or “Polymerized” means the act of, or the resultof, the placement of molecular bridges between molecules or tetramericsubunits where the size and weight of the resulting polymerized moleculeis increased with respect to native or tetrameric hemoglobin.Polymerized hemoglobin is not tetrameric hemoglobin. Polymerization maybe accomplished using various polymerizing agents, includingglutaraldehyde, imido esters, or others, in a biochemically suitablecarrier, as is well known to those skilled in the art.

[0037] “Pyridoxylated” or “pyridoxylation” refers to the method of, orthe result of, binding pyridoxal-5′-phosphate containing molecules to ahemoglobin molecule by reacting the molecule with pyridoxal-5′-phosphate(“P5P”) or 2-Nor-formyl pyridoxal-5′-phosphate. Pyridoxylation has beenshown to favorably alter the reversible oxygen binding capacity, i.e.increase the P₅₀ of certain mammalian hemoglobins, e.g. humanhemoglobin.

[0038] “Stable” or “stability” refer to the state or characteristic ofhemoglobin solutions that are resistant to degradation and have a longershelf life than non-stable solution. For example, hemoglobin solutionthat have been stabilized according to the invention will, compared tosolutions that have not been prepared according to the invention, haveless or slower tetramer elaboration during storage of the solution. Thestability of a hemoglobin solution is dependent on several otherparameters that are independent of tetramer elaboration, including, forexample, how quickly deoxyhemoglobin is converted to oxyhemoglobin ormethemoglobin. This parameter may be controlled by, among other ways,preventing oxygen from entering the packaged solution during storage.Stabilized hemoglobin solutions may still degrade, but do so at a slowerrate than non-stabilized solutions.

[0039] The invention provides a polymerized, hemoglobin solutionessentially free of tetrameric (native or intramolecularly crosslinked)hemoglobin, stromal and various other contaminants. The solution isphysiologically acceptable as well as therapeutically and clinicallyuseful. The product has reversible oxygen binding capacity which isnecessary for oxygen transport properties. The product demonstrates goodoxygen loading and unloading characteristics in usage, which correlatesto having an oxygen-hemoglobin dissociation curve (P₅₀) similar to wholeblood. The product binds oxygen with high affinity in the capillariesthrough the lungs and then adequately releases oxygen to the tissues inthe body. The product also does not require compatibility studies withthe recipient.

[0040] In one aspect, the product may also have a half-life whenadministered to humans of about at least 15 hours. In another aspect,the half life is greater than about 24 hours. The hemoglobin product canbe used to replenish essentially all of a human patient's blood volumewithout causing vasoconstriction, renal toxicity, hemoglobinuria orother problems associated with intravenous administration of syntheticor semisynthetic oxygen carriers and blood substitutes.

[0041] The half-life of the resulting product of the invention isdetermined in vivo in mammals, e.g., humans. Typically, a blood sampleis removed from the mammal a period of time after the mammal has beeninfused with the product. The amount of the product is then determinedby centrifuging the blood sample, expressing the plasma portion,determining plasma hemoglobin levels spectrophotometrically, and thencorrelating the amount of product remaining in the mammal to thehalf-life of the product.

[0042] The method of the invention yields a stabilized polymerizedhemoglobin solution with an enhanced shelf life. Generally, tetramerelaborates as the polymerized hemoglobin solution ages. Elaboratedtetramer can be removed from the aged hemoglobin solution by the processof the invention. Aged hemoglobin solutions that have been processed toremove tetramer exhibit slower tetramer elaboration upon further storageas compared to newly processed (non-aged) hemoglobin solutions. Thus,hemoglobin solutions processed according to the invention exhibit agreater stability with respect to tetramer elaboration than newlyprocessed solutions.

[0043] Generally, it has been found that physiologically and clinicallyuseful hemoglobin solutions contain less than 1.0% tetramer.Accordingly, since tetramer elaborates over time during storage, toallow a reasonable shelf life for the hemoglobin solution, it isdesirable that newly processed hemoglobin solutions contain less thatabout 0.3% tetramer, with the expectation that tetramer in solution willelaborate, but the solution will remain physiologically useful untiltetramer reaches 1.0%. FIG. 1 shows the typical amount of tetramerelaboration of a hemoglobin solution stored at 2-8° C. In a solutionwith beginning tetramer levels of about 0.5%, tetramer levels rise togreater than 1.0% after about 18 months. FIG. 2 shows the typical amountof tetramer elaboration for a hemoglobin solution stored at 23-27° C. Ina solution with a beginning tetramer level of about 0.5%, tetramerlevels rise to above 1.0% in about 3-4 weeks. Thus, storage ofhemoglobin solutions at increased temperatures increases the rate oftetramer elaboration. Aging of tetramer solutions can be accomplishedmerely be allowing the hemoglobin solution to stand for extended periodsof time. Higher temperatures age the hemoglobin solutions faster thanlower temperatures.

[0044]FIG. 3 shows a comparison of the tetramer elaboration between anewly processed hemoglobin solution and an “aged” hemoglobin solutionprocessed according to the invention (“reprocessed”). Both solutionswere stored at 2-8° C. After 24 months, tetramer increased about 0.8% inthe new hemoglobin solutions while, in the reprocessed solution,tetramer increased only about 0.3%. Over 36 months the tetramer in thereprocessed solution increased about 0.4%. Likewise, FIG. 4. shows thattetramer elaborates faster in newly processed solutions as compared toreprocessed solutions when stored at 23-27°.

[0045] In one aspect, the method of the invention involvespre-elaborating the hemoglobin solution to enhances the stability of asolution by heating the solution to accelerate tetramer elaboration. Theelaborated tetramer can be removed from the solution to provide asolution that is more stable with respect to tetramer elaboration thanpolymerized solutions that have not been heat treated to acceleratetetramer elaboration. Once elaborated tetramer has been removed from thehemoglobin solution, further tetramer elaboration is slowed ordecreased.

[0046] Heat treatment may occur either before or after purification ofthe solution. If the solution is heat treated during processing, suchheat treatment should follow polymerization of the solution. Thesolution can then be purified to remove substantially all the tetramer.If heat treated after purification, the solution must be re-purified toremove the elaborated tetramer.

[0047] Heat treating may be accomplished by subjecting the polymerizedhemoglobin solution to about 45-55° C. for about 20-30 hours. It isexpected that other processing temperatures and time will suffice inorder to elaborate tetramer since it has been shown that tetramerelaboration is a function of, among other things, time and temperature.

[0048] The process of the invention provides a further advantage in thatit can render the final product substantially free of microbial andviral antigens and pathogens. Such microbial and viral antigens andpathogens are reduced to non-detectable levels, i.e., the product issterile as determined by the analysis set forth in the United StatesPharmacopoeia, XXIII Chapter 71. Examples of such antigens and pathogensinclude, for example, bacterial, rickettsial, fungal, protozoan, viraland other organisms. Most importantly, the process provides a biologicalproduct free of viruses that cause hepatitis and acquired immunedeficiency syndrome (AIDS).

[0049] Heat treatment of the hemoglobin solution results in thesubstantial inactivation of viruses. Viral inactivation heat treatmentcan be a separate step of heat treatment or may be combined with theheat treatment to remove tetramer. Generally, for the purposes ofproduction operator safety, it is preferred that viral reduction heattreatment be conducted after the hemoglobin has been removed from redblood cells. However, it is expected that heat treatment of the solutionfollowing polymerization will also accomplish the desired viralinactivation. If a pre-polymerization viral reduction heat treatmentstep has been eliminated, temperature of the post-polymerization heattreatment to elaborate tetramer can be increased to about 60-62° C., orother suitable temperature, to ensure viral inactivation. Thus, one heattreating step following polymerization will, in certain aspects,accomplish both viral reduction and tetramer elaboration.

[0050] The biological product of this invention, when infused in amountsof up to at least about 10.0 L, does not cause vasoconstriction, renaltoxicity, hemoglobinuria and other problems implicated with intravenousadministration of known hemoglobin solutions containing physiologicallyundesirable amounts of tetrameric hemoglobin. Intravenous administrationof the product produced by the process described herein results in noappreciable decrease in urine production, no appreciable decrease inglomerular filtration rate, no appreciable extravasation into theperitoneal cavity and no appreciable change in the color of urineproduced.

[0051] Therefore, the process of the invention provides an acellular redblood cell substitute useful in the treatment of trauma, myocardialinfarction, stroke, acute anemia and oxygen deficiency disorders such ashypoxemia, hypoxia or end stage hypoxia due to impairment or failure ofthe lung to fully oxygenate blood. The product also is useful in thetreatment of any disease or medical condition requiring a resuscitativefluid (e.g., trauma, specifically hemorrhagic shock), intravascularvolume expander or exchange transfusion. In addition to medicaltreatment, the product can be useful in preserving organs fortransplants.

[0052] In one aspect, the starting material in the process of theinvention is whole human blood or packed red blood cells. Generally, itis desirable, but not critical, to use source red blood cells that havebeen in storage for no more than 2 weeks past the expiration dateindicated on the blood storage bag. The use of human whole bloodoutdated by more than 2 weeks provides additional difficulty inextracting the hemoglobin and removing cellular remnants such as stromalproteins and contaminants. In addition, the processes described hereinare applicable to all hemoglobins with minor modifications within theskill of the art.

[0053] If human blood is used as a staring material, during red cellaspiration and filtration, the red blood cells (RBC) are asepticallyextracted from donor bags without introducing air into the blood andpassed across a series of filters to result in a RBC suspension havingreduced amounts of leukocytes and platelets. The resulting suspension isthen subjected to cell washing/lysing.

[0054] The suspension is washed under carbon monoxide atmosphere with anabout 1% NaCl solution to remove residual plasma proteins. The washedRBC are then treated with water for injection (“WFI”) to lyse the cellsand the resulting mixture is clarified using a cross flow filtrationunit. Other methods of lysing red blood cells known to those of skill inthe art may be used including, for example, mechanically or sonicallylysing the cells. The clarified product may then be heat-treated forviral inactivation and to precipitate additional stromal material whichis removed by filtration. The product of this procedure is a stroma-freehemoglobin (SFH) solution with a THb of about 3% (w/v).

[0055] Following clarification, the solution containingcarboxyhemoglobin is preferably concentrated and degassed to yield astroma free hemoglobin solution containing deoxyhemoglobin.Degassification involves first saturating the carboxyhemoglobin solutionwith oxygen for about 16 hours to yield a solution of oxygenatedhemoglobin and about 7% by weight, based on the total weight ofhemoglobin, of carboxyhemoglobin. Subsequently, the oxygen is driven offwith nitrogen, argon or helium to form a solution containing freehemoglobin, i.e., uncomplexed hemoglobin, and about 7% by weight, basedon the total weight of hemoglobin, of oxyhemoglobin. The resultingdegassed solution is filtered and transferred into a vessel for chemicalmodification.

[0056] Subsequent to degassification, the stroma-free hemoglobinsolution comprising human hemoglobin should be pyridoxylated usingpyridoxal-5′-phosphate (P5P) at a molar ratio of pyridoxal-5′-phosphateto hemoglobin of about 1:1 to 3:1. Alternatively, the stroma-freehemoglobin may be pyridoxylated using 2-Nor-2 formylpyridoxal-5′-phosphate. A reducing agent such as sodium cyanoborohydrideor preferably sodium borohydride is added to the pyridoxylation mixture.Excess reagents and salts are removed by dialysis against pyrogen freewater or, preferably, diafiltration with WFI. Hemoglobin from sourcesother than human blood may not need pyridoxylation. Those skilled in theart of hemoglobin solutions readily understand when pyridoxylation isrequired.

[0057] The stroma-free, hemoglobin solution is polymerized using anymethod known to those skilled in the art of hemoglobin solutions.Preferably, an aqueous glutaraldehyde is used as a polymerizing agent.The duration of polymerization and the amount of glutaraldehyde added isdependent on volume of the hemoglobin solution, the desired yield ofpolymers and the desired molecular weight distribution. In general,longer polymerization times increase the yield and the molecular weightdistribution of the polymers. A yield of approximately 75% by weight ofpolymers, based on the total weight of hemoglobin, is obtained in about16-18 hours. The preferred end point of the polymerization is defined asthat point where the solution contains about 75% by weight of polymers,based on the total hemoglobin weight, as monitored by size-exclusionHPLC. Alternatively, the endpoint is defined as the point at which thesolution contains about 65% of polymers based on the total weight ofhemoglobin, i.e., about 2.5 hours.

[0058] Following polymerization, the reaction should be quenched withthe appropriate agent. In one aspect, the polymerization reaction isquenched by the addition of aqueous glycine. The glycine should be addedas quickly as possible. The cross-links are then stabilized by adding,again as quickly as possible, a solution of aqueous sodium borohydride.This polymerized solution is subsequently concentrated and thendiafiltered. Water is finally added to the solution until the solutioncontains about 4% by weight hemoglobin.

[0059] In another aspect, the solution may be “hot quenched” by heatingthe solution to 40-50° C. for at least three hours concurrent with theaddition of glycine to drive the quench reaction to completion. FIG. 5shows a comparison of the tetramer elaboration between a newly processedhemoglobin solution and a hemoglobin solution subject to a hot quenchfor three hours. Both solutions were stored at 2-8° C. After 10 months,tetramer increased between 0.4-0.5% in the new hemoglobin solutionwhile, in the hot quenched solution, tetramer increased only about 0.4%.FIG. 6 shows the differences in tetramer elaboration between newlyprocessed solutions and hot quenched solutions when stored at 23-27° C.It is expected stability of the solution would be enhanced by subjectingto the solution to a longer hot quench, for example, up to twenty-fourhours. Higher temperatures, up to about 65° C., may also be employed.

[0060] Polymerization according to the invention results in a high yieldof polymers having a narrow molecular weight range as shown in FIG. 9and the Examples below.

[0061] In another aspect, the post-quench polymerized solution may bepre-elaborated by heat treatment to elaborate tetramer. The heattreatment may be postponed until any point after purification, but thesolution would require repurification. Heat treatment may beaccomplished by heating the solution to above about 45° C. for at leastabout 24 hours. If viral inactivation is desired at this point, thesolution may be heated to above about 60° C. An antioxidant such asascorbic acid may be added to prevent formation of methemoglobin duringthe heat treating process.

[0062]FIG. 7 shows a comparison of the tetramer elaboration between anewly processed hemoglobin solution and a pre-elaborated hemoglobinsolution processed according to the invention. Both solutions werestored at 2-8° C. After 15 months, tetramer increased about 0.5% in thenew hemoglobin solutions while, in the pre-elaborated solution, tetramerincreased only about 0.4%. Likewise, FIG. 8 shows that tetramerelaborates faster in newly processed solutions as compared topre-elaborated solutions when stored at 23-27° C.

[0063] The polymerized, pyridoxylated hemoglobin solution is thenpurified. In one aspect, purification is accomplished under anatmosphere of oxygen to oxygenate the solution utilizing columnchromatography, filtration, e.g., membrane filtration, or both, toremove residual unpolymerized (tetrameric) hemoglobin from the solution.The purified polymerized hemoglobin solution is then concentrated toabout 6% using an ultrafiltration apparatus in preparation for gasexchange.

[0064] The concentrated solution is then deoxygenated with nitrogen. Thedeoxygenation takes place at about 10-12° C. until the amount ofoxyhemoglobin in the solution is less than about 16% by weight of thetotal hemoglobin.

[0065] The resulting deoxygenated, purified, and polymerized hemoglobinsolution is then concentrated by ultrafiltration under a nitrogenatmosphere in a cooled vessel. The pH is adjusted to about 8.8-9.0, andthe amounts of electrolytes may be adjusted as necessary to levelsrepresenting that of normal plasma. In addition, conventionalantioxidants such as glutathione, ascorbate or glucose may be optionallyadded. After the solution is concentrated to the desired level,preferably about 10% by weight total hemoglobin, the solution issterilized by filtration and transferred via a sterile transferapparatus into suitable pharmaceutically acceptable containers.

[0066] If the hemoglobin solution was not previously heat treated tofacilitate tetramer elaboration, the solution may be heat treated andrepurified to remove elaborated tetramer. If antioxidants andformulation chemicals have been added, these may be removed bydiafiltration prior to heat treatment and purification.

[0067] In addition, as an alternative to heat treatment, tetramerelaboration of the finished hemoglobin solution may be accomplished byallowing the solution to age at an appropriate temperature. The solutioncan then be repurified to remove the elaborated tetramer. Generally, itis expected that the solution should age until tetramer levels exceedabout 1-3%, with higher tetramer levels giving increased stabilitybenefits after the solution is purified to remove tetramer. However, itis expected that the advantages of the invention will be accomplished ifthe solution is aged for any period of time, so long as the elaboratedtetramer is removed after aging of the solution.

[0068] The characteristics of the resulting hemoglobin solution areshown in FIG. 9 and are as follows: Total Hemoglobin (g/dl)  9.5-10.5Methemoglobin (% of total Hb) <8.0 Carboxyhemoglobin (% of total Hb)<5.0 P₅₀ (torr) 26-32 Osmolality (mmol/Kg) 280-360 Sodium (mmol/L)135-155 Potassium (mmol/L) 3.5-4.5 Chloride (mmol/L)  85-110 Free Iron(ppm) <2.0 Molecular Wt. Dist. - 128 Kd peak (%)  9-23 Molecular Wt.Dist. - 192 Kd peak (%) 16-18 Molecular Wt. Dist. - 256 Kd peak (%)49-74 Tetramer (64K)(%) ≦1.0 Endotoxin (EU/mL)  <0.03 Phospholipidsng/Hb <50   Glycolipids (ng/Hb) <2  

EXAMPLES

[0069] The following examples demonstrate certain aspects of theinvention. However, it is to be understood that these examples are forillustrative purposes only and do not purport to be wholly definitive asto conditions and scope of this invention. All temperatures areexpressed in degrees Celsius unless otherwise specified. It also shouldbe appreciated that when typical reaction conditions (e.g., temperature,reaction times) have been given, the conditions which are both above andbelow these specific ranges can also be used, though generally lessconveniently.

[0070] Unless noted to the contrary, all vessels and tanks used in theinventive process are made of 316-L Stainless Steel, preferably apharmaceutical grade of such stainless steel that has been highlypolished and therefore easily and rapidly cleaned. The variousconnecting pipes and tubes are made of the same stainless steel or of apharmaceutical grade Teflon or silicone tubing. The filters andmembranes used in the process may be purchased from Millipore Inc.,Pall-Filtron, or Cuno Inc.

[0071] Analytical Size Exclusion Chromatography HPLC according to theinvention is carried out according to the following procedure. Thesample is diluted to 0.2 g/dl with 0.1 M sodium phosphate buffer atabout pH 6.9, filtered through a 0.2μ filter and injected into an HPLCsystem consisting of the following components (in order of system flow):

[0072] 1. Agilent Technologies 1100 Isocratic Pump

[0073] mobile phase is 0.1 M sodium phosphate at about pH 6.9

[0074] flow rate is 0.5 mL/minute

[0075] 2. 45 cm PEEK or titanium tubing, 0.010 in. I.D.

[0076] 3. Agilent 1100 Autosampler

[0077] 4. 18 cm PEEK or titanium tubing, 0.010 in. I.D.

[0078] 5. 0.5μ precolumn filter frit

[0079] 6. 9 cm PEEK or titanium tubing, 0.010 in. I.D.

[0080] 7. Toso TSK G3000SWXL 40×60 mm guard column

[0081] 8. 24 cm PEEK or titanium tubing, 0.010 in. I.D.

[0082] 9. Toso TSK G3000SWXL 300×7.8 mm Analytical column

[0083] 10. 23 cm PEEK or titanium tubing, 0.010 in. I.D.

[0084] 11. Agilent 100 variable wavelength detector.

[0085] wavelength: 280 nm

[0086] flow cell: 14 μL vol., 10 mm pathlength

[0087] range: 2 AUFS

[0088] time constant: 10 seconds

[0089] The peak absorbance at 280 nm is recorded by an AgilentChemstation, which integrates the individual peak areas and calculatesthe total Hemoglobin area for each polymeric species.

Example 1

[0090] Cell Aspiration and Filtration

[0091] Referring now to FIG. 11, donor bags 20 of outdated blood (wholeblood or packed red blood cells) are situated in a suitable asepticaspiration apparatus 22. A needle in the aspiration apparatus puncturesthe donor bag, introduces about 150 ml of a 1% (w/v) aqueous sodiumchloride solution and aspirates the outdated blood from the donor bagunder reduced pressure or vacuum. The aspirated blood is passed throughleukocyte adsorption depth filter 24 or alternatively through two 5μdepth filters in series 26. As the blood passes through the filters,leukocytes are removed from the blood. Typically, about 225 units ofoutdated whole blood are aspirated, filtered and subsequentlytransferred to Tank 1 as shown in FIG. 11. The filters are then rinsedwith about 75 liters of a 1% (w/v) aqueous sodium chloride solution.

Example 2

[0092] Cell Wash and Lysis

[0093] Prior to the introduction of the blood into Tank 1, Tank 1 ischarged with about 40-50 L of a 1% aqueous sodium chloride solution.After all 225 units of outdated whole blood have been aspirated,filtered and transferred, and the filters have been rinsed, the tankcontains about 365-395 liters of a 4% total hemoglobin solution. Duringthe aspiration and filtering steps, Tank 1 is maintained at a reducedpressure, i.e., a vacuum of 20-28 inches Hg. Once all the outdated bloodhas been transferred to Tank 1, the vacuum is switched off and carbonmonoxide is introduced into the tank so that the tank contains anatmosphere of carbon monoxide.

[0094] Tank 1 is coupled to a 0.65μ tangential flow filter 28 as shownin FIG. 11. The initial charge of 365-395 liters of 4% total hemoglobinsolution is concentrated to approximately 215-225 L of a 7% totalhemoglobin solution by microfiltration through the tangential flowfilter. The pH of the hemoglobin solution at this point is about 6 to6.5. Subsequent to concentrating to 7% total hemoglobin, the solution iswashed by adding a 1% (w/v) sodium chloride solution, diafiltering andremoving the filtrate at the same rate sodium chloride solution isadded. The 215-225 L of hemoglobin solution is typically washed withabout 8 volumes of the 1% sodium chloride solution (about 1,800 L).Subsequent to washing, the solution is concentrated to about 90-95 L,i.e., about 16% total hemoglobin, and water for injection (“WFI”) isadded to bring the volume of the solution up to about 220 L. With theaddition of the WFI, the cells swell and rupture releasing hemoglobininto solution. The concentration of the resulting hemoglobin solution isabout 7% total hemoglobin (THb).

[0095] The resulting solution is clarified while still in Tank 1. Thesolution is first concentrated to about 90 L and the filtrate istransferred to Tank 2. As the solution is pumped across the filter, redblood cells, stroma contaminants and cell wall material is retained andremoved by the filter. The remaining 90 L of solution in Tank 1 iswashed (diafiltered) with about 280 L of WFI and the wash is added toTank 2. The material remaining in Tank 1 is then concentrated to about20 L and the filtrate added to Tank 2. The volume resulting in Tank 2 isabout 405-415 L of a 3.3% total hemoglobin solution.

Example 3

[0096] Optional Heat Treatment For Viral Reduction and StromalPrecipitation

[0097] The resulting solution of stroma-free hemoglobin is then heattreated in Tank 2 at a temperature of about 60-62° C. over a period ofabout 10 hours. During this time, the solution is moderately agitated.As the solution is heated and passes a temperature of about 55° C., aprecipitate forms.

Example 4

[0098] Clarification and Viral Reduction

[0099] The resulting 3.3% THb stroma-free, heat treated hemoglobinsolution is then filtered through a 0.2μ prefilter 30 followed by a 0.1μprefilter 32 and then pumped through a 100 kD viral reductionultrafilter (not shown) into Tank 3.

Example 5

[0100] Ultrafiltration/Concentration

[0101] The filtered hemoglobin solution is then concentrated to 85-105 L(about 14% THb) and subsequently washed and diafiltered with about 4volumes of WFI (350 L). The concentration and diafiltration isaccomplished using a 10 kD molecular weight ultrafilter 34. Drain 35associated with ultrafilter 34 collects filtrate. At this point, the 14%total hemoglobin solution contains less than about 50 ng of phospholipidper gram of hemoglobin, less than about 2 ng of glycolipid per gram ofhemoglobin, less than about 1% methemoglobin, less than about 0.03 unitsof endotoxin per milliliter at a pH of about 6 to 6.5. The hemoglobin inthe solution is carboxyhemoglobin.

Example 6

[0102] Degassification

[0103] The resulting carboxyhemoglobin solution is then transferred to a175L vessel (Tank 4) where the carboxyhemoglobin is first oxygenated andthen deoxygenated. Tank 4 is fitted with a gas sparge ring coupled tooxygen and nitrogen gas lines, a feed from the tank bottom to a meteredspray apparatus positioned at the top of Tank 4, and a foam overflowcollector connected to foam can 36 such that foam generated in Tank 4 isfed into foam can 36 where the foam condenses into liquid and is fedback into Tank 4. As an alternative to the foam can 36, Tank 4 can befitted with a mechanical foam breaker. Tank 4 further includes a centermounted, gas dispersion agitator. Foam can 36 includes a gas vent forremoval of gas. The solution in Tank 4 is a 13% total hemoglobinsolution.

[0104] During a first oxygenation step, oxygen is sparged through thesolution at a rate sufficient to have uniform dispersion of gas in thevessel. The vessel is sparged at a rate of about 66 L/min. with gas.Oxygenation of the carboxyhemoglobin is conducted for a period of about18 hours such that the resulting solution contains less than 5%carboxyhemoglobin based on the weight of total hemoglobin. Oxygenationis conducted at a temperature of about 10° C. The foam generated in Tank4 is collected in Foam Can 36 and after settling, the resulting solutionis transferred back into Tank 4.

[0105] After oxygenation, the solution is sparged with a similar flow ofnitrogen for about 3-3.5 hours or until less than 10% oxyhemoglobinbased on the weight of total hemoglobin remains in the solution. Thenitrogen sparge is conducted at a temperature of about 10° C. and a pHof about 6.95-7.10. Alternatively, carboxyhemoglobin could be convertedto deoxyhemoglobin using a membrane exchanger. It is noted that there issubstantially no denaturing of the hemoglobin as would normally beexpected from the foaming step.

Example 7

[0106] Chemical Modification

[0107] Referring now to FIG. 12, the deoxyhemoglobin solution istransferred to Tank 5 for chemical modification. To Tank 5 containingthe deoxyhemoglobin solution at about 4° C. is then added an aqueoussolution of pyridoxyl-5-phosphate (P5P) (93.75 g/L) at a 1:1 to 3:1 P5Pto hemoglobin molar ratio. A 2:1 molar ratio of P5P to hemoglobin ispreferred. The pyridoxylation is conducted at a temperature of about 4°C. The P5P solution is typically added over about 1 minute and mixed forapproximately 15 minutes, after which a sodium borohydride/sodiumhydroxide solution is added to the hemoglobin solution at a molar ratioof sodium borohydride to hemoglobin of about 20:1. A suitable aqueoussodium borohydride/sodium hydroxide solution contains 0.8 g of sodiumhydroxide per 2 liters and 90.8 g of sodium borohydride per 2 liters.The borohydride solution is added as rapidly as possible over a periodof about 1 minute and then stirred for one hour.

Example 8

[0108] Reactant Removal

[0109] The resulting 150 L solution of pyridoxylated hemoglobin issubsequently diafiltered using 10K Dalton ultrafilter 38 to removeexcess reactants with 4 volumes (600 L) of WFI. Drain 40 associated withultrafilter 38 collects the filtrate from filter 38.

Example 9

[0110] Polymerization

[0111] To Tank 5 containing the pyridoxylated hemoglobin is addedsufficient WFI to prepare a 4.5% total hemoglobin solution (about 265 Lof hemoglobin solution). A glutaraldehyde solution is added to thepyridoxylated hemoglobin solution at a molar ratio of glutaraldehyde tohemoglobin of about 24:1. The glutaraldehyde solution is typically addedover a period of about 2.5 hours by a metering pump to the hemoglobinsolution. The polymerization reaction is allowed to proceed for about 18hours. The target molecular weight distribution is about 75% polymer and25% tetramer. The target polymers have molecular weights of less thanabout 600,000 with a predominant fraction of the molecular weightsresiding in the 100,000-350,000 range.

[0112] When the polymerization reaction reaches the target molecularweight distribution (after about 18 hours), aqueous glycine (about 166g/L) is added (as a quench) to the hemoglobin solution at a 140:1 molarratio of glycine to hemoglobin. At this point, the solution may beheated to 40-50° C. for at least three hours to drive the quenchreaction to completion (“hot quench”).

[0113]FIG. 10 shows an HPLC tracing of the resulting polymerized,glycine-quenched hemoglobin product. The resulting solution is thenmixed for about 10 minutes after which a sodium borohydridesodium/hydroxide solution (having the concentration identified above) isadded to the hemoglobin solution at a 28:1 molar ratio of sodiumborohydride to hemoglobin. This resulting mixture is stirred for about 1hour. The solution is then concentrated to about 150 L (ultrafilter 38)and washed with 4 volumes (600 L) of WFI. An additional aliquot ofsodium borohydride at the same molar ratio as indicated above is addedto the concentrated solution and again mixed for 1 hour. The resultingsolution is washed with 4 volumes of WFI (600 L).

Example 10

[0114] Optional Heat Treatment for Tetramer Elaboration

[0115] At this point, the solution may be heat treated to elaboratetetramer. The solution may be subject to heating at 45-55° C. for about20-30 hours. If the viral reduction is desired during this step, thetemperature may be increased to above 60° C. To heat the solution, aheating medium, such as a propylene glycol solution at about 80° C., iscirculated through the tank jacket while vigorously agitating the Hbsolution. After heating, the hemoglobin solution is cooled to about 2-8°C.

Example 11

[0116] Purification

[0117] The resulting solution is oxygenated by allowing the solution tostand under an oxygen atmosphere and is subsequently transferred to apurification system 42. The purification may be achieved by columnchromatography, filtration, preferably membrane filtration(diafiltration), or a combination of filtration and columnchromatography.

[0118] In one embodiment, the solution is transferred to chromatographyfeed vessel, Tank 6, as shown in FIG. 13. In this embodiment, theresulting solution of oxyhemoglobin is then diluted to about 200 L (4%total hemoglobin) in Tank 6 and the concentration of chloride isadjusted to 22 mM with sodium chloride solution. No adjustment of sodiumconcentration is necessary.

[0119] Five 40 L aliquots of the resulting hemoglobin solution are thenchromatographed using Column 44. Column 44 contains an affinity gelwhich is an agarose gel modified with a yellow dye (commerciallyavailable from Affinity Chromatography, Ltd., as Mimetic Yellow No. 1)having greater affinity for polymer than tetramer.

[0120] The chromatography is accomplished as follows. 40 L ofoxygenated, polymerized, pyridoxylated, stroma-free hemoglobin solutionis loaded onto Column 44. The column is washed with 15 column volumes(about 750 L) of 30 mM aqueous NaCl buffer to remove tetramer. Thecolumn is then washed with about 250 L of a 300 mM sodium chloridebuffer to wash the polymer off. Polymer fractions are collected in Tank7. Unwanted fractions are sent to drain 46. After each aliquot isremoved, the column is regenerated with 15 mM HCL solution (150 L),re-equilibrated with 30 mM aqueous NaCl (250 L) and another aliquot offeed solution (40 L) is loaded to the column. The column is again washedwith 30 MM NaCl followed by 300 mM NaCl. 40 L aliquots of hemoglobinsolution are added to the column and chromatographed until Tank 6 isempty.

[0121] The collected fractions in Tank 7 are ultrafiltered(concentrated) using filter 48 associated with drain 50 to a volume ofabout 40 L (6% total hemoglobin). The concentrated hemoglobin solutionis then transferred to gas exchange Tank 8 for deoxygenation.

[0122] Alternatively, the solution is transferred to a filtrationrecycle vessel 10, as shown in FIG. 14. The hemoglobin is then dilutedto about 4% THb in Tank 10. The 4% THb solution is then diafilteredusing 10 mM NaCl and a 300,000 molecular weight filter 52 commerciallyavailable from Millipore Corporation. The filtration is continued untilabout 97% of the hemoglobin material passes through the filter and intoTank 11. (About 3% of the material, i.e., high molecular weightpolymers, is retained in Tank 10). The amount of hemoglobin isdetermined spectrophotometrically using a cooximeter.

[0123] The resulting material in Tank 11 is about 4-8% THb and containsabout 7-10% tetramer based on THb. The 4-8% THb is then diafilteredusing 10 mM NaCl and a 100,000 molecular weight filter 54 commerciallyavailable from Pall-Filtron associated with drain or trap 56. Thefiltration is continued until the level of tetramer, as determined bysize exclusion chromatography using a Toso BioSep 300×7.8 mm column, isless than 1.0% of the hemoglobin mass by weight. The resulting purifiedhemoglobin solution remains initially in Tank 11 and is subsequentlytransferred to gas exchange Tank 8 for deoxygenation.

Example 12

[0124] Deoxygenation

[0125] Gas exchange Tank 8 may be the same tank as Tank 4 or,preferably, a different tank. Gas exchange Tank 8 is equipped inessentially the same fashion as gas exchange Tank 4 and is attached tofoam can 58 or equipped with a mechanical foam breaker in a fashionidentical to that of Tank 4. Deoxygenation is accomplished in about 2.5hours with a nitrogen sparge at about 10° C. and a solution pH of about8.8. Nitrogen sparging is continued until less than about 16%oxyhemoglobin, based on the weight of total hemoglobin, remains in thesolution. The resulting deoxyhemoglobin solution is subsequentlytransferred to Tank 9 for formulation.

[0126] If the heat treatment step of Example 10 has been postponed, itmay be conducted at this stage. If heat treatment is conducted now, thepurification procedures of Examples 11 and the deoxygenation proceduresof this Example should be repeated. The heat treatment step of Example10 and the heat treatment step at this point are optional and may beeliminated completely.

Example 13

[0127] Formulation

[0128] In formulation Tank 9, the solution is first concentrated toabout 7% total hemoglobin, and the pH is adjusted to about 8.8 to 9.0 at4° C. The pH is adjusted using 0.2 M NaOH. Glucose and glycine are addedto achieve final concentrations of about 1 g/L and 3.5 g/L respectively.Potassium chloride is added to the solution to obtain a potassiumconcentration of about 3.5 to 4.5 mM. 3 M sodium chloride is then addedto obtain an 85-110 mM chloride concentration. Sodium lactate issubsequently added to obtain a 135-155 mM concentration of sodium ion.Finally, a 0.45 molar ascorbic acid solution is added until the ascorbicacid concentration reaches about 1000 mg/L. The pH is adjusted to8.7-9.1 at 10-15° C. using 0.22 M NaOH. The resulting hemoglobinsolution has a final osmolality of about 280-360 mmole per kg.

[0129] The formulated hemoglobin solution is then concentrated to about10% total hemoglobin using filter 60 associated with trap 62. The 10%hemoglobin solution is then sterilized by filtration through filter 64and aseptically filled into presterilized bags.

Example 14

[0130] Solution Pre-Elaboration

[0131] Whether or not the solution has been subjected to the hot quenchor optional heat treatment steps to elaborate tetramer duringprocessing, the solution may be heated anytime after formulation toelaborate tetramer by heating the solution to about 45-55° C. for about20-30 hours, or until tetramer has increased above about 1-3%. Followingheat treatment the solution should be purified as in Example 11 anddegassed as in Example 13. Since the solution at this point has alreadybeen formulated, the additives introduced in Example 12 are preferablyremoved by diafiltration prior to purification, and then reintroducedduring re-formulation.

Example 15

[0132] Re-processing Aged Hemoglobin Solutions

[0133] Out of date or aged hemoglobin solutions may be reprocessed as inExample 14, without heat treating. Typically, hemoglobin solutions thatare older than about 12-18 months at storage conditions of about 2-8° C.have tetramer levels of greater than about 1.0%. This material may beintroduced to Tank 5 and purified and reformulated according to Examples11-13. Since the solution at this point has already been formulated, theadditives introduced in Example 13 are preferably removed bydiafiltration prior to purification, and then reintroduced duringre-formulation.

[0134] In the foregoing, there has been provided a detailed descriptionof preferred embodiments of the invention for the purpose ofillustration and not limitation. It is to be understood that all othermodifications, ramifications and equivalents obvious to those havingskill in the art based on this disclosure are intended to be within thescope of the invention as claimed.

What is claimed is:
 1. A method for producing a substantially tetramerfree hemoglobin solution comprising: a) polymerizing hemoglobin insolution; b) heat treating the polymerized hemoglobin in solution; c)removing tetramer from the polymerized hemoglobin in solution.
 2. Themethod of claim 1 wherein the hemoglobin is derived from mammalianblood.
 3. The method of claim 2 wherein the mammalian blood is humanblood and the hemoglobin is pyridoxylated.
 4. The method of claim 1wherein the hemoglobin is derived from bovine blood.
 5. The method ofclaim 1 wherein the hemoglobin is polymerized with glutaraldehyde. 6.The method of claim 1 wherein the tetramer is removed by filtration. 7.The method of claim 1 wherein the heat treatment comprises heating thesolution above about 45° C. for at least about 24 hours.
 8. The methodof claim 1 wherein the tetramer concentration at the completion of step(c) is less than about 1.0% of total hemoglobin in the solution.
 9. Themethod of claim 1 wherein the tetramer concentration at the completionof step (c) is less than about 0.3% of total hemoglobin in the solution.10. The method of claim 1 further comprising, in addition to step (c),removing tetramer from the solution prior to the heat treating.
 11. Themethod of claim 10 wherein tetramer is removed from the solution priorto the heat treating until the solution is essentially tetramer free.12. The method of claim 11 wherein the tetramer concentration prior tothe heat treating is less than about 1.0% of total hemoglobin in thesolution.
 13. The method of claim 12 wherein the tetramer concentrationprior to the heat treating is less than about 0.3% of total hemoglobinin the solution.
 14. A hemoglobin solution produced by the method ofclaim
 1. 15. A method for stabilizing an essentially tetramer freepolymerized hemoglobin solution comprising treating the polymerizedhemoglobin solution to partially degrade the polymerized hemoglobin totetramer and removing the tetramer from the solution.
 16. The method ofclaim 15 wherein the treating comprises aging the solution.
 17. Themethod of claim 15 wherein the treating comprises aging the solutionuntil the tetramer concentration is above about 1.0% of the totalhemoglobin in solution.
 18. The method of claim 15 wherein the treatingcomprises heating the solution.
 19. The method of claim 15 wherein thetreating comprises heating the solution until the tetramer concentrationis above about 1.0% of the total hemoglobin in solution.
 20. The methodof claim 15 wherein the hemoglobin is derived from mammalian blood. 21.The method of claim 15 wherein the mammalian blood is human blood andthe hemoglobin is pyridoxylated.
 22. The method of claim 15 wherein thehemoglobin is derived from bovine blood.
 23. The method of claim 15wherein the hemoglobin is polymerized with glutaraldehyde.
 24. Themethod of claim 15 wherein the tetramer is removed by filtration. 25.The method of claim 18 wherein the heating comprises heating thesolution above about 45° C. for at least about 24 hours.
 26. A methodfor producing a stabilized, polymerized hemoglobin solution comprising:a) producing a polymerized hemoglobin solution; b) removing tetramerfrom the polymerized hemoglobin solution to produce a substantiallytetramer free polymerized hemoglobin solution; c) aging the polymerizedhemoglobin solution; and d) removing the elaborated tetramer.
 27. Themethod of claim 26 wherein the aging comprises storing the hemoglobinsolution until the tetramer concentration is greater than about 1.0% oftotal hemoglobin.
 28. The method of claim 26 wherein the aging comprisesstoring the hemoglobin solution until the tetramer concentration isgreater than about 3.0% of total hemoglobin.
 29. The method of claim 26wherein the aging comprises storing the hemoglobin solution for longerthan one year.
 30. A method for producing a substantially tetramer freehemoglobin solution comprising: e) subjecting hemoglobin in solution toa polymerization reaction comprising a polymerizing agent; f) quenchingthe polymerization reaction with a quenching agent; g) heating thesolution during the quenching; h) removing tetramer from the polymerizedhemoglobin in solution.
 31. The method of claim 30 wherein the solutionis heated to at least about 40° C. during the quenching for at leastthree hours.
 32. The method of claim 31 wherein the solution is heat toabout 40-50° C.